The movement of charged molecules in an electric field, known as electrophoresis, is the basis of several separation techniques utilized in the separation of large organic macromolecules, such as for example proteins, RNA, DNA and the like. The basis of all the above mentioned technique consists of applying a potential drop across a medium containing the charged molecules. Depending on the charge carried by the molecule, it will be driven toward the electrode of opposite sign relative to that carried by the molecule. Many macromolecules are zwitterions, having charged side chains on their surface which can be either positively or negatively charged. At some particular value of pH, the sum of the positive charges will balance the negative charges and the molecule will be neutral. This value of pH, which is unique for each molecule, is referred to as the isoelectric point, or pI, of the molecule. At this pH, the molecule, being neutral, is immobile in the electric field. At other pH values the molecule will have a mobility dependent on its charge, which in turn depends on the pH, thus the mobility of the molecule will be a function of solution pH. If a molecule is in a solution having a pH &gt; pI, the charge and mobility of the molecule will be negative while for the case when the molecule is in a solution having a pH &lt; pI, its charge and mobility will be positive.
The existence of a unique pI value for macromolecules provides the basis for isoelectric focusing. In this technique, a pH gradient is established in the medium and the molecule, on being driven through this gradient by the electric field, when it reaches the pH equal to its pI value, becomes electrically neutral and stops moving. The usual procedure is to first form the pH gradient by electrophoresing a mixture of carrier polyampholytes through the medium in which the separation is to be effected. These polyampholytes or carrier ampholytes are typically small, mobile, multi-charged polymers and which preferably exhibit a manifold of different pI values spanning the entire pH range. The pH gradient arises due to a concentration gradient which forms in response to the applied electric field. Thus, for the ionic species to achieve an equilibrium condition in the electric potential gradient, a concentration gradient forms which balances the applied field. The smaller and more mobile the ionic species in the medium of interest, the more rapidly the pH gradient will be established. The substance containing the species to be separated is then introduced into the medium whereupon the component species are separated by the action of the electric field. In light of the discussion above, the mobility of the molecule will be a function of its position in the pH gradient. Therefore, because the mobility and charge of a molecule changes sign on either side of the pI of the molecule, when present in a pH gradient with an applied field, the molecule will be driven towards the position in the cell (and there neutralized) having a pH equal to the pI of the molecule, regardless of its initial position or charge in the cell.
The medium for electrophoresis can be either a free electrolyte, porous paper or a gel. A general problem in scaling up any of the electrophoresis techniques for the separation of macromolecules in a free electrolyte is ohmic heating of the electrolyte. Typically, solutions which stabilize molecules such as proteins are usually highly electrically conducting, being respectable ionic conductors, with the result that high current densities are obtained which cause ohmic heating of the electrolyte. This heating sets up convection currents within the electrolyte which act to disrupt the moving boundaries as well as preventing the accumulation of the neutralized proteins in the cell positions having a pH corresponding to their pI values. One method of dealing with this problem is to add a convection suppression component to the medium, such as a chemically inert and electrically neutral species which, when present in sufficient concentration, acts as a convection suppression medium. A drawback to this approach is that an extra purification step is required to separate the purified material from the convection suppression agent. Accordingly, there is a need to provide an electrochemical cell having a structure and geometry which provide a substantially convection free region thereby permitting the efficient separation and isolation of products purified in this way.
In the gel electrophoresis of proteins and other macromolecules, the gel is employed for two main purposes; the first being to suppress convection currents arising due to temperature variations in the medium arising from ohmic heating as pointed out above, and the second being to provide a molecular sieve for aiding in separating the molecules on the basis of size as well as charge. This is possible since the pore size of the gel can be accurately controlled during polymerization of the latter. As with the scale-up of free electrophoresis, the scale up of gel electrophoresis is not practical. While a very powerful and reliable way of accurately separating macromolecules with very similar pI values, gel electrophoresis is only practically and economically capable of separating and producing very small amounts of a pure material, typically nanogram to microgram quantities. Part of the reason for this is the expense of the carrier polyampholytes required to set up the pH gradient. Another reason for the low yields is that in gel electrophoresis, once the gel medium is set up the material to be separated can be applied only at the boundaries of the gel which severely limits the amount of material which can be purified. Accordingly, there is a need to provide a method and apparatus for the large scale industrial separation of materials on the basis of their isoelectric points.